Diabetes mellitus poses a major health threat in the United States and developing world. This Pilot & Feasibility proposal investigates the application of total protein synthesis by native ligation to diabetes research. We anticipate that the proposed studies will facilitate structural analysis of diabetes-related gene products and provide novel reagents for proteomics. Two years of support are sought to demonstrate the promise of this approach. Aim 1 focusses on the insulin signaling pathway in the nematode C. elegans. This pathway controls aging and life span, in part through regulation of the dauer state. The C. elegans genome contains 34 putative insulin-like genes and a single orthologue of the human insulin receptor (designated daf-2). Because the insulin-like sequences are highly divergent from mammalian insulins, their classification is controversial. We propose to (a) synthesize representative C. elegans polypeptides, (b) determine their structures, and (c) characterize their biochemical function (binding to and regulation of the daf-2 tyrosine kinase receptor). The insulin signaling pathway in C. elegans promises an opportunity to combine powerful genetic, biochemical, and structural tools in a tractable model organism. Aim 2 focuses on a newly described mammalian hormone, resistin. Secreted by adipocytes, this polypeptide blocks insulin action and thus is implicated in the clinical link between human obesity and Type II diabetes mellitus. We propose to (a) synthesize resistin, (b) determine its structure, (c) characterize structure-function relationships by scanning mutagenesis, and (d) create novel reagents for cloning the putative resistin receptor. The proposed studies have translational potential as a basis for new human therapeutics. In summary, rapid and large-scale synthesis of novel cysteine-rich gene products by native peptide ligation promises to expedite structural and functional analysis of diabetes-related regulatory pathways. The proposed pilot studies will dissect an insulin signaling pathway in a genetic model of senescence and aging (Aim 1) and a novel mechanism of metabolic homeostasis and insulin resistance in human obesity.